Cloning And Expression Of CdMMP-12

Matrix metalloproteinases (MMPs) have long been associated with cancer-cellbehavior.Matrix metalloproteinases (MMPs) can regulate the tumourmicroenvironment, and their expression and activation is increased in almost allhuman cancers compared with normal tissue. MMPs are proteolytic enzymesand their basic mechanism of action — degradation of proteins — regulatesvarious cell behaviours with relevance for cancer biology. These includecancer-cell growth, differentiation, apoptosis, migration and invasion, and theregulation of tumour angiogenesis and immune surveillance. Humanmacrophage elastase (HME;MMP-12) was first identified as an elastolyticmetalloproteinase secreted by inflammatory macrophages 30 years ago.MMP-12 shares many features typical of MMPs, including its domain structure.MMP-12 is secreted as a 54-kd pro-form protein that undergoes self-activationthrough autolytic processing to produce 45-and 22-kd active forms of theenzyme. The major substrate for MMP-12 is elastin, which is abundant in thelung and arterial wall.Based on the human MMP-12 gene, I designed a new MMP-12 gene, which isused in optimal codons.So the recombinate plasmid express more protein thanbefore. The signal peptide at the N-termination can lead the protein to theperiplasm. Six histidines at the C-termination will let the purification easy.First we splicing these primers designed with optimal codons into a new DNAfragment, then cut it by EcoRI and XhoI at the termination. At the same time,the plamid has been cut in the same way. These two fragment purified by kit canbe connected directly in the coupled reaction, then transformated into the E.coli.When the recombinant plamid was constructed, we analysis its total coprotein todetermine if the recombinant protein express. We found the optimal temperatureis 25℃ and optimal induce time is 4 hours. Furthermore, the protein isolated inthe periplasm can be purified by nickel ion affinity chromatography.